Background
Methods
Isolates
Faecal specimens
SpeeDx Carba (beta) PCR
In-house PCR
Set | Forward primer (5′-3′) | Reverse primer (5′-3′) | Probe | Reference |
---|---|---|---|---|
KPCa | GGCCGCCGTGCAATAC | GCCGCCCAACTCCTTCA | FAM-TGATAACGCCGCCGCCAATTTGT-BHQ1 | [18] |
NDMa | GACCGCCCAGATCCTCAA | CGCGACCGGCAGGTT | VIC-TGGATCAAGCAGGAGAT-MGB-NFQ | [18] |
OXA-48-like | GTAGCAAAGGAATGGCAA | CCTTGCTGCTTATTcTCAc | FAM-TCC(+A)GA(+G)CA(+C)AA(+C)TACG-Dabcyl | [19] |
IMP-4-likeb | GGCAGTATTTCCTCTCATTT | GCAGCTCATTAGTTAATTCAG | FAM-CATAGTGACAGCACGGGCGGAAT-BHQ1 | [20] |
VIMb | CGCGGAGATTGAGAAGCAAA | AGCCGCCCGAAGGACATC | HEX-TTGGACTTCCTGTAACGCGTGCA-BHQ1 | [20] |
16S rRNAa | TGGAGCATGTGGTTTAATTCGA | TGCGGGACTTAACCCAACA | Quasar670-CACGAGCTGACGACARCCATGCA-BHQ2 | [18] |
PCR controls and environmental practices
Detection limit comparisons
DNA sanger sequencing
Set | Forward primer (5′-3′) | Reverse primer (5′-3′) | Amplicon size (bp) |
---|---|---|---|
OXA-48-like | CCTCGATTTGGGCGTGGTTA | TCCGATGTGGGCATATCCAT | 475 |
IMP-4-like | GAAGGTGTTTATGTTCATACTTCGT | TCACTGTGACTTGGAACAACCAGTTTTGC | 557 |
VIM | GAGTTGCTTTTGATTGATACAGC | TCGGTCGAATGCGCAGCACC | 291 |
Results
Isolate extracts
Faecal specimens
n | Carba (beta) assay Ct values (range; mean) | In-house assays Ct values (range; mean) | Sequencing result | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
KPC | NDM | OXA-48-like | IMP-4-like/VIM | IC | KPC | NDM | OXA-48-like | IMP-4-like | VIM | 16S rRNA | ||
1 | neg | neg | neg | POSITIVE (25.4) | POSITIVE (22.6) | n/a | n/a | n/a | POSITIVE (31.4) | neg | POSITIVE (11.2) | IMP-4 |
1 | neg | neg | POSITIVE (27.0) | neg | POSITIVE (23.4) | n/a | n/a | neg | n/a | n/a | POSITIVE (15.5) | OXA-181 |
6 | neg | neg | POSITIVE (25.1–28.9; 27.1) | neg | POSITIVE (22.3–24.4; 23.0) | n/a | n/a | neg | n/a | n/a | POSITIVE (12.4–18.7; 14.2) | n/a |
452 | neg | neg | neg | neg | POSITIVE (21.4–28.7; 22.8) | n/a | n/a | n/a | n/a | n/a | n/a | n/a |